Neisseria gonorrhoeae (Ng) is the etiologic agent of gonorrhea. With over 100 million annual cases worldwide, gonorrhea is one of the most common sexually transmitted diseases. In recent years, an alarming increase in antibiotic resistance among Neisseria gonorrhoeae strains has been a cause for great concern among all national and international health agencies. The current emergence of cephalosporin-resistant strains (so far cephalosporins are our last resort antibiotic treatment for gonorrhea) around the world emphasizes the need for both new treatments for gonorrhea, as well as a better understanding of the basic mechanisms behind antibiotic resistance. As with many other bacteria, Ng bacteria are rarely found as single cells. Instead they are first found in small clusters or microcolonies of a few tens to a few thousands bacteria and then in fully formed biofilms of up to millions of cells. Bacteria within biofilms are more resistant to antibiotic treatment than their free-living counterparts. Biofilms also play a major role in pathogen colonization and disease. While much progress has been made in understanding the architecture of the mature biofilm, the process by which biofilms develop remains less well understood. Understanding the early stages of biofilm development may lead to new therapies and treatments of important human diseases. Similarly to what has been shown between human cells, we postulate that the impact of physical forces is crucial in the formation and architecture of Ng microcolonies, the precursors of Ng biofilms. A key element in the formation of Ng microcolonies is the Type IV pilus (Tfp). Tfp are retractable bacterial fibers involved in many aspects of Ng physiology including motility, adhesion, infection, DNA uptake and biofilm formation. We have shown that the retraction of Tfp from Ng microcolonies can exert forces in the nanoNewton range (approximately 100,000 times the bodyweight of a single bacteria) and that these forces are capable of triggering signaling events in human cells. The objective of this study is to discover how the extreme forces of Tfp retraction shape Ng microcolony formation and the behavior of Ng bacteria within these communities. We will obtain this objective by pursuing three aims: identify how Tfp retraction forces shape microcolonies, determine the role of Tfp retraction forces in the initial steps of biofilm differentiation, and determine the role ofTfp retraction forces in the resistance to antibiotics and the spreading of antibiotic resistance. This work is of particular significance as it addresses a largely unknown and important stage of biofilm development. The findings here will lay the groundwork for future research on an important topic of human health. The proposed project is innovative as it will, for the first time, assess the role of physical forces in the formation and function of Ng microcolonies and achieve this at the single cell level.